Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
1999-07-12
2003-09-23
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C436S518000, C436S537000, C436S538000, C436S018000, C436S164000, C436S166000, C436S177000, C436S805000, C436S824000, C435S007100, C435S967000, C435S007920, C435S007930, C435S007940, C435S007950
Reexamination Certificate
active
06623982
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the fields of bioaffinity separations and diagnostic testing of biological samples. More specifically, the invention provides compositions and methods which, may be used in magnetic separation assays and enrichment procedures for controlling endogenous magnetic particle aggregation factors which, if uncontrolled, would obscure visualization of isolated entities. Also provided are methods for constructing and synthesizing reversible aggregation factors and the resulting compositions which simultaneously enhance recovery of rare biological substances while facilitating observation of substances so isolated.
BACKGROUND OF THE INVENTION
Several publications are referenced in this application by numerals in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Many laboratory and clinical procedures employ bio-specific affinity reactions. Such reactions are commonly utilized in diagnostic testing of biological samples, or for the separation of a wide range of target substances, especially biological entities such as cell, viruses, proteins, nucleic acids and the like. Various methods are available for analyzing or separating the above-mentioned target substances based upon complex formation between the substance of interest and another substance to which the target substance specifically binds. Separation of complexes from unbound material may be accomplished gravitationally, e.g. by settling, or, alternatively, by centrifugation of finely divided particles or beads coupled to the target substance. If desired, such particles or beads may be made magnetic to facilitate the bound/free separation step. Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions. See, for example, U.S. Pat. No. 4,554,088 and
Immunoassavs for Clinical Chemistry
, pp. 147-162, Hunter et al. eds., Churchill Livingston, Edinborough (1983). Generally, any material which facilitates magnetic or gravitational separation may be employed for this purpose. However, in the past 20 years the superiority of magnetics for performing such separations has led to its use in many applications.
Magnetic particles generally fall into two broad categories. The first category includes particles that are permanently magnetizable, or ferromagnetic. The second category comprises particles that demonstrate bulk magnetic behavior only when subjected to a magnetic field. The latter are referred to as magnetically responsive particles. Materials displaying magnetically responsive behavior are sometimes described as superparamagnetic. However, materials exhibiting bulk ferromagnetic properties, e.g., magnetic iron oxide, may be characterized as superparamagnetic only when provided in crystals of about 30 nm or less in diameter. Larger crystals of ferromagnetic materials, by contrast, retain permanent magnet characteristics after exposure to a magnetic field and tend to aggregate thereafter due to strong particle-particle interactions. Magnetic particles can be classified as large (1.5 to about 50 microns), small (0.7-1.5 microns), and colloidal or nanoparticles (<200 nm). The latter are also called ferrofluids or ferrofluid-like and have many of the properties of classical ferrofluids. Liberti et al pp 777-790, E. Pelizzetti (ed) “Fine Particles Science and Technology” Kluwer Acad. Publishers, Netherlands, 1996.
Small magnetic particles are quite useful in analyses involving bio-specific affinity reactions, as they are conveniently coated with biofunctional polymers (e.g., proteins), provide very high surface areas and give reasonable reaction kinetics. Magnetic particles ranging from 0.7-1.5 microns have been described in the patent literature, including, by way of example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of these particles are disclosed to be useful solid supports for immunologic reagents.
In addition to the small magnetic particles mentioned above, there are a class of large magnetic particles ranging in size from approximately 1.5-50 microns, which also have superparamagnetic behavior. Typical of such materials are those invented by Ugelstad (U.S. Pat. No. 4,654,267) and manufactured by Dynal, (Oslo, Norway). The Ugelstad process involves the synthesis of polymer particles which are caused to swell and magnetite crystals are embedded in the swelled particles. Other materials in the same size range are prepared by synthesizing the particle in the presence of dispersed magnetite crystals. This results in the trapping of magnetite crystals in a polymer matrix, thus making the resultant materials magnetic. In both cases, the resultant particles have superparamagnetic behavior, which is manifested by the ability to disperse readily upon removal of the magnetic field. Unlike magnetic colloids or nanoparticles, these materials, as well as small magnetic particles, are readily separated with simple laboratory magnetics because of the mass of magnetic material per particle. Thus, separations are effected in gradients from as low as a few hundred gauss/cm on up to about 1.5 kilogauss/cm. Colloidal magnetic particles, (below approximately 200 nm),on the other hand, require substantially higher magnetic gradients because of their diffusion energy, small magnetic mass per particle and Stokes drag. U.S. Pat. No. 4,795,698 to Owen et al. relates to polymer-coated, colloidal, superparamagnetic particles. Such particles are manufactured by precipitation of a magnetic species in the presence of a biofunctional polymer. The structure of the resulting particles, referred to herein as single-shot particles, has been found to be a micro-agglomerate in which one or more ferromagnetic crystallites having a diameter of 5-10 nm are embedded within a polymer body having a diameter on the order of 50 nm. The resulting particles exhibit an appreciable tendency to remain in aqueous suspension for observation periods as long as several months. U.S. Pat. No. 4,452,773 to Molday describes a material similar in properties to those described in Owen et al., which is produced by forming magnetite and other iron oxides from Fe
+2
/Fe
+3
via base addition in the presence of very high concentrations of dextran. Materials so produced have colloidal properties and have proved to be very useful in cell separation. This technology has been commercialized by Miltenyi Biotec, Bergisch Gladbach, Germany.
Another method for producing superparamagnetic colloidal particles is described in U.S. Pat. No. 5,597,531. In contrast to the particles described in the Owen et al. patent, these latter particles are produced by directly coating a biofunctional polymer onto pre-formed superparamagnetic crystals which have been dispersed, e.g., by sonic energy into quasi-stable crystalline clusters ranging in size from about 25-120 nm. The resulting particles, referred to herein as direct coated (DC) particles, exhibit a significantly larger magnetic moment than Owen et al. or Molday nanoparticles of the same overall size and can be separated effectively in magnetic gradients greater than about 6 kGauss/cm.
Magnetic separation techniques are known wherein a magnetic field is applied to a fluid medium in order to separate ferromagnetic bodies from the fluid medium. In contrast, the tendency of colloidal superparamagnetic particles to remain in suspension, in conjunction with their relatively weak magnetic responsiveness, requires the use of high-gradient magnetic separation (HGMS) techniques in order to separate such particles from a fluid medium in which they are suspended. In HGMS systems, the gradient of the magnetic field, i.e., the spatial derivative, exerts a greater influence upon the behavior of the suspended particles than is exerted by the strength of the field at a given point. High gradient magnetic separation is useful for separating a wide variety of magnetically l
Liberti Paul A.
Rao Galla Candra
Terstappen Leon W. M. M.
Chin Christopher L.
Dann Dorfman Herrell and Skillman
Do Pensee T.
Immunivest Corporation
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